Biologically applicable water-soluble heterogeneous catalysts for para-hydrogen induced polarization

ABSTRACT

A heterogeneous catalyst composition for para-hydrogen induced polarization includes ligand-capped nanoparticles dispersed in water. The ligand-capped nanoparticles include metal nanoparticles that are surface functionalized with organic ligands, a molecular weight of the organic ligands is no greater than 300 g/mol, and the organic ligands each includes multiple binding moieties as coordinates sites for binding to a nanoparticle surface.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.15/799,498, filed Oct. 31, 2017; which claims the benefit of U.S.Provisional Application No. 62/415,986, filed Nov. 1, 2016, the contentof each of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

This disclosure generally relates to heterogeneous catalysts that arewater-soluble and can yield high polarization of substrate molecules.

BACKGROUND

Nuclear magnetic resonance (NMR) hyperpolarization aims to overcome alow signal of NMR by increasing spin polarization up to four orders ofmagnitude compared to thermal polarizations from state-of-the-artsuperconducting magnets. Hyperpolarization techniques include spinexchange optical pumping, the more established technique of dynamicnuclear polarization (DNP), and the use of para-hydrogen inducedpolarization (PHIP) or Signal Amplification by Reversible Exchange(SABRE). Polarization techniques depend on the context and application.Each technique can potentially lead to the development of promisingcontrast agents for biomedical imaging, with an example of dissolutionDNP for which in vivo human use has been demonstrated. To generatehyperpolarization using PHIP or SABRE, a para-enriched spin state ofhydrogen is first created by passing hydrogen over a catalyst at lowtemperatures, generating close to 100% para-state below about 25 K. Thenearly pure stable singlet spin state of para-hydrogen can subsequentlybe utilized to hyperpolarize a molecule of interest by an additionreaction or by a catalyst-mediated, reversible exchange process.

PHIP hyperpolarized substrates have been proposed as contrast agents forangiography or cancer detection. A major drawback is that the generationof sufficiently high nuclear spin polarization in biocompatible solventsis obtained using a homogeneous catalyst. However, homogeneous catalystsraise biotoxicity concerns, since the homogeneous catalysts cannot bereadily separated from a solution. Without sufficient separation, ahyperpolarized molecule is mixed with a homogeneous catalyst, and thepresence of the catalyst in solution can pose toxicity issues,particularly if heavy metals are used, such as rhodium. Although apreliminary study has shown that a state-of-the-art homogeneous catalystproduces subclinical hepatic and renal toxicity in rats, further studiesremain to clarify toxicity concerns. To address such concerns,approaches are desired in which a catalyst can be separated from apotential molecular imaging agent. One such approach is a phaseseparation technique in which a nuclear-spin polarization is generatedin water followed by extraction of a substrate of interest. Theextraction process, however, represents a stage during which thegenerated polarization inevitably decays and a loss in polarizationresults. Attempts have been made to develop heterogeneous catalysts, butthese typically operate in non-biocompatible solvents, yield lowpolarizations in water, and suffer from leaching problems whereimmobilized ligands leach in solution.

It is against this background that a need arose to develop theembodiments described herein.

SUMMARY

Embodiments of this disclosure are directed to nanoparticle-basedcatalysts that are water-soluble and can yield high polarization ofsubstrate molecules via PHIP. In some embodiments, cysteine-cappedplatinum nanoparticles serve as a heterogeneous catalyst which yieldslarge polarization by PHIP in water. The use of water as a solventpromotes biocompatibility. Moreover, the use of a heterogeneous catalystalso promotes biocompatibility: ligand-capped nanoparticles can beimmobilized on a solid support, and the nanoparticles also can befiltered using, for example, a filtration membrane. Ligand-cappednanoparticles of embodiments of this disclosure can be used to generatenuclear-spin polarized molecules for in vivo biological applications,such as medical imaging applications for magnetic resonance imaging(MRI) detection.

Advantageously, ligand-capped nanoparticles of embodiments of thisdisclosure can be used to generate high polarization under conditionsthat favor biocompatibility and water solubility. Specifically, aselection of ligands renders the nanoparticles water-soluble ordispersible in water. Also, a relatively high surface coverage of thenanoparticles by the ligands favors pair-wise hydrogenation, which is acondition for generating strong nuclear-spin polarization by PHIP.Further, the use of the ligand-capped nanoparticles as a heterogeneouscatalyst addresses a separation issue: polarized molecules can bereadily separated from the heterogeneous catalyst through a filtrationstage to remove the catalyst. As another option, the nanoparticles canbe immobilized on a solid support (e.g., silica or alumina), and thesolid support can remain in a reactor and does not flow with a liquidphase. As a further option, the nanoparticles can be bound to largersized water-soluble or dispersible particles. For example, the largersized particles can be polystyrene beads (or other polymeric beads)having sizes of about 500 nm and which are dispersible in water or heavywater. More generally, the larger sized particles can have sizes (e.g.,in terms of a diameter or another lateral dimension) in a range of about200 nm to about 10 μm, about 200 nm to about 5 μm, or about 200 nm toabout 1 μm.

In an aspect according to some embodiments, a heterogeneous catalystcomposition for PHIP includes nanoparticles that are capped or surfacefunctionalized with ligands.

In some embodiments, the nanoparticles are metal nanoparticles which areformed of, or include, a metal, such as a platinum group metal(platinum, rhodium, ruthenium, iridium, osmium, or palladium), oranother transition metal. Metal nanoparticles which are formed of, orinclude, a combination of two or more different metals, such as in theform of an alloy or a mixture, are also encompassed by this disclosure.In some embodiments, the nanoparticles have sizes (e.g., in terms of adiameter or another lateral dimension) in a range of about 0.5 nm toabout 100 nm, about 0.5 nm to about 80 nm, about 0.5 nm to about 50 nm,about 0.5 nm to about 30 nm, about 0.5 nm to about 10 nm, or about 0.5nm to about 5 nm. In some embodiments, at least one of the nanoparticleshas a size (e.g., in terms of a diameter or another lateral dimension)in a range of about 0.5 nm to about 100 nm, about 0.5 nm to about 80 nm,about 0.5 nm to about 50 nm, about 0.5 nm to about 30 nm, about 0.5 nmto about 10 nm, or about 0.5 nm to about 5 nm. In some embodiments, thenanoparticles have an average size (e.g., in terms of an averagediameter or another lateral dimension) in a range of about 0.5 nm toabout 100 nm, about 0.5 nm to about 80 nm, about 0.5 nm to about 50 nm,about 0.5 nm to about 30 nm, about 0.5 nm to about 10 nm, about 0.8 nmto about 10 nm, about 0.5 nm to about 5 nm, about 0.8 nm to about 5 nm,about 0.8 nm to about 4 nm, about 0.8 nm to about 3 nm, about 0.8 nm toabout 2.5 nm, or about 0.8 nm to about 2 nm. In some embodiments, thenanoparticles are generally spherical or spheroidal with aspect ratiosof about 3 or less, about 2 or less, or about 1.5 or less, althoughnanostructures having other shapes and aspect ratios are alsoencompassed by this disclosure, such as aspect ratios greater than about3.

In some embodiments, the ligands are organic ligands. In someembodiments, the ligands are homogeneous with respect to one another,although the use of different ligands is also encompassed by thisdisclosure. In some embodiments, the ligands are hydrophilic, and eachincludes at least one hydrophilic moiety. Examples of suitablehydrophilic moieties include carboxyl group, hydroxyl group, carbonylgroup, sulfhydryl (or thiol) group, amino group, phosphate group, andcharged forms thereof. In some embodiments, the ligands each includesmultiple (e.g., two or more) binding moieties as coordinates sites forbinding to a nanoparticle surface. Examples of suitable binding moietiesinclude sulfhydryl (or thiol) group, amino group, and charged formsthereof. In some embodiments, the ligands each includes sulfhydryl (orthiol) group and amino group as binding moieties, and further includesat least one hydrophilic moiety. In some embodiments, the ligands are,or include, amino acids. In some embodiments, the ligands each consistsof, or consists essentially of, an amino acid. In some embodiments, theligands are, or include, cysteine, such as L-cysteine.

In some embodiments, the composition includes a weight content of theligands of at least about 5 wt. % relative to a combined weight of thenanoparticles and the ligands, such as about 8 wt. % or greater, about10 wt. % or greater, about 12 wt. % or greater, about 14 wt. % orgreater, or about 16 wt. % or greater, and up to about 24 wt. % orgreater. In some embodiments, a surface coverage of the nanoparticles bythe ligands is at least about 4.2 ligands per nm², such as about 4.4ligands per nm² or higher, about 4.6 ligands per nm² or higher, about4.8 ligands per nm² or higher, about 5 ligands per nm² or higher, about5.5 ligands per nm² or higher, about 6 ligands per nm² or higher, about6.5 ligands per nm² or higher, about 7 ligands per nm² or higher, about7.5 ligands per nm² or higher, or about 8 ligands per nm² or higher, andup to about 8.4 ligands per nm² or higher. In some embodiments, asurface coverage of the nanoparticles by the ligands is at least about0.8 mol/g in terms of moles of the ligands relative to a combined weightof the nanoparticles and the ligands, such as about 0.85 mol/g orhigher, about 0.9 mol/g or higher, about 0.95 mol/g or higher, about 1mol/g or higher, about 1.05 mol/g or higher, about 1.1 mol/g or higher,about 1.15 mol/g or higher, about 1.2 mol/g or higher, about 1.25 mol/gor higher, about 1.3 mol/g or higher, or about 1.35 mol/g or higher, andup to about 1.5 mol/g or higher. In some embodiments, a molecular weightof the ligands is no greater than about 300 g/mol, such as about 280g/mol or less, about 250 g/mol or less, about 230 g/mol or less, about200 g/mol or less, about 180 g/mol or less, about 150 g/mol or less, orabout 130 g/mol or less.

In some embodiments, the composition includes a solvent, and theligand-capped nanoparticles are dispersed in the solvent. In someembodiments, the solvent is water. In some embodiments, the solvent isheavy water or D₂O. In some embodiments, a concentration of theligand-capped nanoparticles is in a range of about 1 mg/mL to about 20mg/mL, about 2 mg/mL to about 15 mg/mL, about 5 mg/mL to about 15 mg/mL,or about 10 mg/mL. In some embodiments, the composition serves as areagent for medical imaging. In some embodiments, an organic substrate(including substrate molecules) is also included, either in a samecontainer or compartment along with the ligand-capped nanoparticlesdispersed in the solvent, or in a separate container or compartment of amedical imaging kit. In some embodiments, the organic substrate is, orincludes, hydroxyethyl acrylate.

In another aspect according to some embodiments, a method for medicalimaging includes combining ligand-capped nanoparticles and an organicsubstrate in a solvent, followed by hydrogenation and polarization ofthe organic substrate, in the presence of the ligand-cappednanoparticles, by flowing or pressurizing para-hydrogen gas, followed byremoving the ligand-capped nanoparticles by filtration or in anothermanner, and followed by conveying the polarized organic substrate to apatient for medical imaging, such as in vivo MM.

In a further aspect according to some embodiments, a method forsynthesis of ligand-capped nanoparticles includes combining ametal-containing precursor and ligands in a solvent, followed byreduction of the metal-containing precursor in the presence of areducing agent. In some embodiments, the metal-containing precursor is aplatinum group metal-containing precursor, such as hexachloroplatinicacid hexahydrate, and the ligands are, or include, cysteine, such asL-cysteine. In some embodiments, the reducing agent is, or includes,sodium borohydride. In some embodiments, a molar ratio of themetal-containing precursor to the ligands is in a range of about 1:2 toabout 2:1, about 1:2 to about 1.5:1, about 1:1.5 to about 1.5:1, orabout 1:1.

Other aspects and embodiments of this disclosure are also contemplated.The foregoing summary and the following detailed description are notmeant to restrict this disclosure to any particular embodiment but aremerely meant to describe some embodiments of this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the nature and objects of some embodimentsof this disclosure, reference should be made to the following detaileddescription taken in conjunction with the accompanying drawing.

FIG. 1. (a) Para-hydrogen (circles with arrows represent hydrogen withquantum correlation) that adsorbs onto a surface of a bare platinumnanoparticle is prone to randomization processes leading to the loss ofquantum correlation. (b) If ligands are attached to the surface of thenanoparticle (wavy lines), the randomization can be reduced, resultingin preservation of the singlet state between the two protons. (c) As aresult of the preserved singlet state on the surface of the cappednanoparticle, para-hydrogen can be used to generate hyperpolarizedproducts in water.

FIG. 2. (a) Reaction of hydroxyethyl acrylate (HEA) to hydroxyethylpropionate (HEP) utilizing para-hydrogen. (b) Hyperpolarized ¹H NMRspectrum shown in absolute values at B₀=about 14.1 T. (c) Thermalpolarized ¹H NMR spectrum after the polarization at B₀=about 14.1 T. Thesignal enhancement in the shown spectrum accounts for =about 145, whichcorresponds to a polarization of P=about 0.7%.

FIG. 3. (a) Schematic of cysteine coordination to a platinum particle.(b) Schematic of acetylcysteine coordination to a platinum particle. Dueto a higher amount of coordination in Cys@Pt, less randomization processof the para-spin state of hydrogen on the particles surface can occur,leading to higher polarization.

FIG. 4. (a) Transmission electron microscopy (TEM) image of Cys1@Ptparticles. (b) TEM image of Cys1.1@Pt particles. (c) TEM image of NAC@Ptparticles. (d) Ultraviolet/visible (UV/vis) absorbance in water ofhexachloroplatinic acid (top curve at origin), Cys1.1@Pt (2^(nd) topmostcurve at origin), NAC@Pt (3^(rd) topmost curve at origin) and Cys1@Pt(bottom curve at origin). The absence of an absorbance peak at about 260nm for the nanoparticle suspensions shows that substantially no platinumions are present to catalyze a reaction.

FIG. 5. Nanoparticle size distribution of NAC@Pt.

FIG. 6. Nanoparticle size distribution of Cys1@Pt.

FIG. 7. Nanoparticle size distribution of Cys1.1@Pt.

FIG. 8. Thermogravimetric analysis of NAC@Pt.

FIG. 9. Thermogravimetric analysis of Cys1 @Pt.

FIG. 10. Thermogravimetric analysis of Cys1.1@Pt.

FIG. 11. ¹H NMR spectra of (a) NAC@Pt and (b) N-acetylcysteine both inD₂O. The line broadening in (a) shows that the ligand is coordinated tothe particles' surfaces.

FIG. 12. ¹H NMR spectra of (a) Cys1@Pt, (b) Cys1.1@Pt and (c) cysteinein D₂O. The line broadening in (a) and (b) shows that the ligand iscoordinated to the particles' surfaces.

FIG. 13. Mercury poisoning test of NAC@Pt. Without mercury the reactionprogresses (squares) whereas the reaction stops upon addition of mercury(circles).

FIG. 14. Mercury poisoning test of Cys1 @Pt. Without mercury thereaction progresses (squares) whereas the reaction stops upon additionof mercury (circles).

FIG. 15. Mercury poisoning test of Cys1.1@Pt. Without mercury thereaction progresses (squares) whereas the reaction stops upon additionof mercury (circles).

FIG. 16. Polarization as a function of particle concentration: NAC@Pt(left bars), Cys1@Pt (middle bars) and Cys1.1@Pt (right bars).

FIG. 17. Thermal polarized signal of ¹³C enriched HEP after 256 scans(top curve) and hyperpolarized signal (bottom curve, ten timesmagnified).

FIG. 18. Particle recycling experiments with Cys1.1@Pt (invertedtriangles), Cys1@Pt (circles) and NAC@Pt (squares). The added linesrepresent the average polarization values achieved with each particle.

FIG. 19. a)¹H spectrum of a product in a supernatant after separatingNAC@Pt and the product in solution at B₀=about 14.1 T. b)¹H spectrum ofthe hyperpolarization experiment after additional HEA was added. Thespectrum shows that no hyperpolarization pattern is observable and thethermal magnetization as not fully built up yet. Thus, significantleaching of the catalyst is not observable.

FIG. 20. a) ¹H spectrum of a product in a supernatant after separatingCys1@Pt and the product in solution at B₀=about 14.1 T. b) ¹H spectrumof the hyperpolarization experiment after additional HEA was added. Thespectrum shows that no hyperpolarization pattern is observable and thethermal magnetization as not fully built up yet. Thus, significantleaching of the catalyst is not observable.

FIG. 21. a) ¹H spectrum of a product in a supernatant after separatingCys1.1@Pt and the product in solution at B₀=about 14.1 T. b) ¹H spectrumof the hyperpolarization experiment after additional HEA was added. Thespectrum shows that no hyperpolarization pattern is observable and thethermal magnetization as not fully built up yet. Thus, significantleaching of the catalyst is not observable.

DETAILED DESCRIPTION

To address biotoxicity concerns associated with the use of homogeneouscatalysts and polarization decay associated with an extraction processfollowing generation of polarization in organic solvents, the use ofheterogeneous PHIP or SABRE catalyst is a desirable strategy to generatea highly pure substrate, since the catalyst can be readily filtered orimmobilized to avoid contamination. For in vivo applications the use ofbiocompatible solvents, such as water in combination with aheterogeneous catalyst is desirable. For example, glutathione-cappedplatinum nanoparticles dispersed in water can be used to generatehyperpolarization. These glutathione-capped platinum nanoparticlesprovide a heterogeneous PHIP catalyst in water yielding significantpolarization of dissolved molecules that remain in a liquid phase;however, measured levels of proton polarization of hydroxyethylpropionate (HEP) are still relatively low (e.g., P=about 0.25%) comparedto the levels desired for in vivo applications (e.g., P>about 1%).Higher polarization can be achieved by capped nanoparticles based on aninsight that the mobility of hydrogen atoms is reduced by ligands,thereby favoring a pair-wise addition mechanism (see FIG. 1).

In accordance with some embodiments of this disclose, cysteine-cappedplatinum nanoparticles are presented as substantially improved catalystsyielding average proton polarizations of P=about 0.65% (or higher) inwater. In comparison, levels of P=1.26% are achieved with a homogeneouscatalyst performing a same experiment using similar conditions andsetup. Thus, the heterogeneous catalyst is competitive withstate-of-the-art homogeneous catalyst for hyperpolarization in water. Ina recycling experiment, it is demonstrated that these nanoparticles canbe used 5 times (or more) without noticeable loss of polarization. It isfurther established that the properties of the ligands for capping thenanoparticles and the nanoparticles' surface coverage are particularfactors for achieving high levels of polarization; indeed, these factorscan be more important than achieving small particle sizes.

In some embodiments, two types of nanoparticles investigated aresynthesized based on a platinum core capped with L-cysteine (Cys), andN-acetyl-L-cysteine (NAC) ligands, respectively. For the synthesis,hexachloroplatinic acid hexahydrate and the desired ligand weresuspended in water and reduced with sodium borohydride, yieldingligand-capped nanoparticles (Scheme 1 a,b). In order to achievenanoparticles with narrow size distributions, with N-acetyl-L-cysteine,a metal-containing precursor to ligand molar ratio of about 1:1.3 wasused (NAC@Pt) and for L-cysteine, the molar ratio was about 1:1 or about1:1.1 (Cys1@Pt and Cys1.1@Pt). Depending on the cysteine concentration,particles with two different average sizes were isolated: about 2.4 nmand about 1.4 nm. The average size for NAC@Pt was found to be about 1.9nm as confirmed by transmission electron microscopy (TEM) (see Examplesection).

In order to confirm the removal of residual platinum ions originatingfrom the hexachloroplatinic acid, two experiments were conducted withall three nanoparticles: (1) Dilute samples with nanoparticles in waterwere characterized by ultraviolet/visible (UV/vis) spectroscopy andcompared to the signal of hexachloroplatinic acid. For platinum ions, acharacteristic absorbance of about 260 nm can be observed, which is thecase for hexachloroplatinic acid but not for any of the synthesizednanoparticles. (2) A mercury poisoning experiment was performed in whichthe hydrogenation of hydroxyethyl acrylate (HEA) was initiated inseparate experiments with the three different particles. Upon additionof mercury the hydrogenation stopped, establishing that thenanoparticles are catalyzing the reaction and not residual platinumions. The ligand binding on the particles was validated by ¹H NMR,resulting in significant dipolar broadening of the ligands' resonances(see Example section). Furthermore, for all of the particle species, aligand coverage of about 16 wt. % was confirmed by thermogravimetricanalysis. Thus, substantially the same amount of platinum catalyzes areaction if hyperpolarization experiments are performed with identicalconcentrations. Hyperpolarization experiments were conducted under inertgas with about 10 mg/mL particle concentrations, about 2 mg HEA andabout 5 bar para-hydrogen in water at about 80° C. Hyperpolarized HEPshows two characteristic lines in the ¹H NMR spectrum at about 2.5 ppmand about 1.0 ppm (FIG. 2). On average, a polarization of P=about 0.65%was observed for both of the Cys@Pt particles, and a polarization ofP=about 0.10% was achieved for NAC@Pt, in contrast to glutathione-cappedplatinum nanoparticles (GSH@Pt) reaching P=about 0.25%. A homogeneouscatalyst is used under identical conditions and a proton polarization ofabout 1.26% is obtained, merely a factor of about two greater than thepolarization achieved with the Cys@Pt particles. Typical conversions inthe proton experiments were about 1% of the starting material. Thisexperiment is, however, not optimized and the use of automaticpolarizers should improve the conversion. Polarization transferexperiments from ¹H to ¹³C nuclei with a custom-built polarizer led toabout 10-fold (P=about 0.01%) signal enhancements and a conversion ofabout 50% in an about 3 s experiment. It is noted that this specificpolarizer design was not optimized for heterogeneous experiments (seeExample section). ¹³C polarizations above about 50% of HEP can beachieved with automated polarizers utilizing a homogeneous catalyst. Asthe ¹H polarization is about twice as high as for the Cys@Ptnanoparticles, it is extrapolated that about 10-25% polarization shouldbe attainable in an optimized device. This amount of polarization issufficient for in vivo experiments. Consequently, the investigatedparticles can serve as a heterogeneous alternative to homogeneouscatalysts.

Particle characteristics are summarized in Table 1. Due to the generallyspherical shape of the nanoparticles, a cubo-octahedral structure can beassumed for which the amount of surface atoms can be estimated. Theamount of ligands covering the surface was found to be the highest forCys1@Pt, followed by NAC@Pt, Cys1.1@Pt and GSH@Pt. However, cysteine hastwo potential coordination sites to a particle's surface: a thiol groupand an amino group. NAC is a derivative of cysteine with the amino groupprotected; thus, the amino group does not coordinate to the particle'ssurface and just the thiol group binds to the surface to stabilize theparticle (FIG. 3). Other infrared investigations on GSH- andcysteine-capped platinum nanoparticles of larger size revealed that uponthe ligands' interaction with the particle surface N—H and S—Hvibrations vanish, which can be understood as an indication for binding.Overall, the cysteine ligands with higher coordination restrict therandomization processes of para-hydrogen on the particles' surfaces. Asa result, the cysteine particles compared to acetylcysteine have about6.5-fold increase in polarization. Other results for another dualcoordination ligand, glutathione-capped platinum particles (GSH@Pt),support this interpretation. The ligand coverage of GSH@Pt by weightpercentage is lower than NAC@Pt but still yields a higher polarization.With respect to the polarization, an increase can occur if fewerplatinum sites are available on the particles' surfaces. Regarding theturnover frequency (TOF) of HEA and hydrogen at about 80° C. and about 1atm hydrogen pressure, it was found that of the synthesized particlesCys1 @Pt show the highest reactivity, which is followed by Cys1.1@Pt andNAC@Pt. For the higher amount of ligands, a higher reaction rate isachieved and para-hydrogen can react faster with HEA. However, GSH@Ptshows a higher catalytic activity than the synthesized particles,although the achieved polarization is lower. GSH on a nanoparticlesurface may allow for a better access for reactants to the surface dueto their packing properties. A better interaction with the metal surfaceexplains the higher conversion but may indicate that more randomizationcan occur due to the higher degree of hydrogen diffusion. Thus, a lossin polarization can be observed, which makes the Cys@Pt particlessuperior alternatives to generate polarization.

TABLE 1 Summary of particle characteristics. Ligands/ Ligands/ Diameter/P/ Ligands/nm² surface atoms/ TOF/

Particle wt % mol/g nm % (sphere) per particle (80° C.) NAC@Pt 16 0.981.9 ± 0.4 0.1 4.8 138 3.0 Cys1@Pt 16 1.32 2.4 ± 0.5 0.65 8.4 256 31.8Cys1.1@Pt 16 1.32 1.4 ± 0.3 0.65 4.7 67 11.0 GSH@Pt

23 0.75 2.0 0.25 4.1 155 87.8

indicates data missing or illegible when filed

A major advantage of heterogeneous catalysts over homogeneous catalystsis their recyclability. Particles can be filtered and reused if anachieved polarization remains substantially constant following repeatedexperiments. In order to test for the particles' recyclability, aparticle concentration of about 15 mg/mL was used to producehyperpolarized HEA five times. After each stage, the particles werecentrifuged, and the supernatant solvent was removed. Subsequently, theparticles were re-suspended in water and re-used to generatehyperpolarized HEP. For all three synthesized particles, thepolarization measured was reproduced in consecutive experiments (seeExample section).

In conclusion, synthesis is performed for two types of nanoparticlesthat are dispersible in water and highly effective in inducinghyperpolarization from para-hydrogen. Due to the optimization of ligandcoverage and ligand-particle interaction, an increase in HEPpolarization greater than about 2-fold was achieved in water compared toGSH@Pt particles. Experiments with two different sized nanoparticlesindicate that the ligand coordinated to the particle's surface may playa more important role in generating hyperpolarization than the particlesize itself. PHIP itself may therefore provide a method to investigatesurface properties of ligand-capped nanoparticles. Additionally, therecyclability of the particles was successfully demonstrated throughfive consecutive uses without noticeable loss in polarization strength.Since the ¹H polarization obtained with the state-of-the-art homogeneouscatalyst is just a factor of about 2 higher than the polarizationachieved for the synthesized particles, the cysteine-based particlesrepresent heterogeneous alternatives; their desired ability to mitigatetoxicity issues makes them desirable candidates for a variety ofclinical molecular imaging applications.

Example

The following example describes specific aspects of some embodiments ofthis disclosure to illustrate and provide a description for those ofordinary skill in the art. The example should not be construed aslimiting this disclosure, as the example merely provides specificmethodology useful in understanding and practicing some embodiments ofthis disclosure.

Materials and Methods

Chemicals

Hexachloroplatinic acid, sodium borohydride, absolute ethanol, cysteineand acetylcysteine were purchased from Sigma-Aldrich. D₂O was purchasedfrom Cambridge isotopes and all chemicals were used without furtherpurification.

Methods for the Particle Characterization

NMR spectra were recorded on a Bruker AV600 or on a Bruker 94/20 Biospecwith about 9.4 T. Transmission electron microscopy (TEM) was performedon a FEI Tecnai T12. Thermogravimetric analysis (TGA) was conductedusing a Perkin Elmer Pyris Diamond TG/DTA from about 25° C. to about650° C. UV/vis characterization was performed on an Agilent 8453 UV-visspectrophotometer.

Synthetic Procedures

Unless otherwise noted, each synthetic stage was performed under inertgas atmosphere.

Synthesis of Acetylcysteine-Capped Platinum Nanoparticles (NAC@Pt)

About 98.4 mg (about 0.19 mmol) of hexachloroplatinic acid hexahydrateand about 40.3 mg (about 0.25 mmol) of acetylcysteine were dissolved inabout 25 mL deoxygenated ultrapure water under argon atmosphere andstirred for about 30 minutes. Subsequently, about 74 mg (about 1.95mmol) NaBH₄ dissolved in about 3 mL ultrapure water was added over about1 minute under argon atmosphere. The brown suspension was stirred forabout an additional hour, for about 3 hours under static vacuum andconcentrated to near dryness. Particles were precipitated with about 30mL deoxygenated, absolute ethanol. After about 30 minutes the ethanolwas decanted and the particles dried under vacuum. Alternatively, theparticles were centrifuged for about 30 minutes and the supernatantdecanted followed by drying under vacuum. Further purification can beachieved by re-suspending the particles in water, centrifuging thesuspension at about 109000 rpm for about 5 hours in an ultracentrifugeand removing the supernatant solvent followed by drying in vacuum.

Synthesis of Cysteine-Capped Platinum Nanoparticles (Cys@Pt)

About 98.4 mg (about 0.19 mmol) of hexachloroplatinic acid hexahydrateand either about 23.0 mg (about 0.19 mmol) or about 25.3 mg (about 0.21mmol) of L-cysteine were suspended in about 25 mL deoxygenated ultrapurewater under argon atmosphere and stirred for about 30 minutes.Subsequently, about 74 mg (about 1.95 mmol) NaBH₄ dissolved in about 3mL ultrapure water was added over about 1 minute under argon atmosphere.The brown suspension was stirred for about an additional hour, for about3 hours under static vacuum and concentrated to near dryness. Particleswere precipitated with about 30 mL deoxygenated, absolute ethanol. Afterabout 30 minutes the ethanol was decanted and the particles dried undervacuum. Alternatively, the particles were centrifuged for about 30minutes and the supernatant decanted followed by drying under vacuum. Ifa platinum precursor to cysteine molar ratio of about 1:1 was used,Cys1@Pt platinum particles were yielded and if a molar ratio of about1:1.1 was used, Cys1.1@Pt particles were obtained.

Characterization of the Nanoparticles

Particle Distribution and UV/Vis

FIG. 4 shows TEM images of Cys1@Pt particles, Cys1.1@Pt particles, andNAC@Pt particles, and UV/vis characterization of the particles.

Particle size distributions were determined for the three differentparticles. For NAC@Pt, Cys1@Pt and Cys1.1@Pt, 169, 187 and 134 particleswere chosen at random. Sizes for the different particles of 1.9±0.4 nm,2.4±0.5 nm and 1.4±0.3 nm were found. The given deviation corresponds tothe standard deviation derived from a Gaussian fit over the particlesize distribution. Histograms of the size distributions are shown inFIGS. 5-7.

Thermogravimetric Analysis (TGA)

Thermogravimetric analysis was performed from about 25° C. to about 650°C. under argon. The TGA curves for the three particles are depicted inFIGS. 8-10 and show that the surfaces of all the particles are about 16wt. % covered.

NMR of the Particles

In order to demonstrate that binding of the ligands to the particles hasoccurred, ¹H NMR spectra were recorded at B₀=about 14.1 T. A significantdipolar broadening of the peaks was observed due to the coordination tothe nanoparticles for all three samples (FIGS. 11-12).

Mercury Poisoning

A mercury poisoning test was performed for the nanoparticles. In thisexperiment, added mercury forms amalgams with the platinum nanoparticleswhich results in catalytic deactivation. If the reaction is catalyzed byplatinum ions rather than the particles, the reaction would progresssince no deactivated amalgam is formed. For the test about 2.0 mg ofparticles were suspended in about 4 mL of water that contained aninternal potassium acetate standard and about 0.4 mL hydroxyethylacrylate was added. Half of the solution was used for the poisoning testand half of it was used to run a control experiment without mercury. Inboth solutions, hydrogen was bubbled over about 60 minutes with a flowrate of about 100 mL/minute. Into one reaction about 20 μL of mercurywas added after about 15 minutes resulting in termination of thehydrogenation, whereas hydrogenation in the control experimentprogresses (See FIGS. 13-15). Thus the hydrogenation is catalyzed by thenanoparticles and not residual platinum ions.

Para-Hydrogen Experiments

¹H NMR experiments with para-hydrogen (about 95% para-enriched) wereperformed on a Bruker AV600 spectrometer (B₀=about 14.1 T). Samples wereprepared in about 5 mm Young tubes from New Era that have been testedfor blind activity before usage. Under inert gas about 2 mg hydroxyethylacrylate (about 0.04 mmol) and various nanoparticle concentrations weresuspended in about 0.5 mL D₂O. Each sample was heated to about 80° C.,pressurized with about 5 bars of para-hydrogen, shaken in the earth'smagnetic field (ALTADENA experiment) and transported into the center ofthe magnet within about 5 s. The spectrum was recorded in a single scan(45°-pulse). After the hyperpolarization experiment, a spectrum wasrecorded with the formed product in thermal equilibrium, and the signalenhancement and the corresponding polarization calculated. Variousnanoparticle concentrations were used to determine the optimal particleconcentration to generate the highest possible polarization and it wasfound to be about 10 mg/mL for all of the tested particles (FIG. 16).Each experiment was repeated at least 3 times. ¹³C polarizationexperiments were performed on a custom-built polarizer at Cedars-SinaiMedical Center according to procedures at about 60° C. As before, the¹³C polarization achieved with the described polarizer was a factor ofabout 10 higher than in thermal equilibrium. FIG. 17 compares thehyperpolarized signal with a signal in thermal equilibrium after 256scans, showing that with the current polarizer setup no significant ¹³Cpolarization could be achieved. Therefore, the construction of anoptimized polarizer in conjunction with a heterogeneous catalyst isdesirable.

Particle Recycling

A further proof that the hydrogenation and thus hyperpolarization iscatalyzed due to the nanoparticles is a recycling experiment. In thisexperiment about 15 mg of particles were used to hyperpolarize HEP atabout 80° C. and the hyperpolarized signal detected. Afterwards theparticles were separated from the supernatant including the hydrogenatedproduct in two different ways: either by centrifuging the particles inD₂O with about 109000 rotations per minute (rpm) for about 2 hours in anultracentrifuge, or by removing the solvent in vacuum followed bywashing the particles with ethanol and drying them under vacuum again.Each of these experiments was performed twice for each type of particle.Afterwards the particles were reused up to four times following the sameprocedure. Over the course of the five performed experiments nosignificant loss in polarization was observed (FIG. 18). To ensure thatno significant amount of platinum has leached into the supernatant orthat no significant platinum ions dissolved in the solvent were causingthe hyperpolarized signal, hyperpolarization experiments with thesupernatant solvent were performed. To the supernatant from thepolarization experiments, about 2 mg HEA were added and ahyperpolarization experiment performed as above was conducted. FIGS.19-21 show that no hyperpolarized signal could be observed. Therefore,it can be concluded that the polarization was indeed generated due tothe platinum nanoparticle catalyst and that no significant leaching hasoccurred.

As used herein, the singular terms “a,” “an,” and “the” may includeplural referents unless the context clearly dictates otherwise. Thus,for example, reference to an object may include multiple objects unlessthe context clearly dictates otherwise.

As used herein, the term “set” refers to a collection of one or moreobjects. Thus, for example, a set of objects can include a single objector multiple objects.

As used herein, the terms “substantially” and “about” are used todescribe and account for small variations. When used in conjunction withan event or circumstance, the terms can refer to instances in which theevent or circumstance occurs precisely as well as instances in which theevent or circumstance occurs to a close approximation. For example, whenused in conjunction with a numerical value, the terms can refer to arange of variation of less than or equal to ±10% of that numericalvalue, such as less than or equal to ±5%, less than or equal to ±4%,less than or equal to ±3%, less than or equal to ±2%, less than or equalto ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, orless than or equal to ±0.05%. For example, a first numerical value canbe “substantially” or “about” the same as a second numerical value ifthe first numerical value is within a range of variation of less than orequal to ±10% of the second numerical value, such as less than or equalto ±5%, less than or equal to ±4%, less than or equal to ±3%, less thanor equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%,less than or equal to ±0.1%, or less than or equal to ±0.05%.

Additionally, amounts, ratios, and other numerical values are sometimespresented herein in a range format. It is to be understood that suchrange format is used for convenience and brevity and should beunderstood flexibly to include numerical values explicitly specified aslimits of a range, but also to include all individual numerical valuesor sub-ranges encompassed within that range as if each numerical valueand sub-range is explicitly specified. For example, a range of about 1to about 200 should be understood to include the explicitly recitedlimits of about 1 and about 200, but also to include individual valuessuch as about 2, about 3, and about 4, and sub-ranges such as about 10to about 50, about 20 to about 100, and so forth.

While the disclosure has been described with reference to the specificembodiments thereof, it should be understood by those skilled in the artthat various changes may be made and equivalents may be substitutedwithout departing from the true spirit and scope of the disclosure asdefined by the appended claims. In addition, many modifications may bemade to adapt a particular situation, material, composition of matter,method, operation or operations, to the objective, spirit and scope ofthe disclosure. All such modifications are intended to be within thescope of the claims appended hereto. In particular, while certainmethods may have been described with reference to particular operationsperformed in a particular order, it will be understood that theseoperations may be combined, sub-divided, or re-ordered to form anequivalent method without departing from the teachings of thedisclosure. Accordingly, unless specifically indicated herein, the orderand grouping of the operations are not a limitation of the disclosure.

1-20. (canceled)
 21. A method for inducing hyperpolarization frompara-hydrogen, comprising: hydrogenating a substrate, in the presence ofligand-capped nanoparticles and a solvent, by flowing or pressurizingpara-hydrogen gas to yield a polarized substrate; and removing theligand-capped nanoparticles from the polarized substrate, wherein theligand-capped nanoparticles comprise metal nanoparticles that aresurface-functionalized with organic ligands, a molecular weight of theorganic ligands is no greater than 300 g/mol, and the organic ligandseach includes multiple binding moieties as coordinating sites forbinding to a nanoparticle surface.
 22. The method of claim 21, whereinthe organic ligands comprise amino acids.
 23. The method of claim 22,wherein the organic ligands comprise cysteine or glutathione.
 24. Themethod of claim 23, wherein the organic ligands comprise cysteine. 25.The method of claim 21, wherein the metal nanoparticles comprise one ormore platinum-group metals.
 26. The method of claim 25, wherein themetal nanoparticles comprise platinum, palladium, ruthenium, rhodium, orany combination thereof.
 27. The method of claim 21, wherein themolecular weight of the organic ligands is no greater than 250 g/mol.28. The method of claim 21, wherein a concentration of the ligand-cappednanoparticles in the solvent is in a range of 1 mg/mL to 20 mg/mL. 29.The method of claim 21, wherein a surface coverage of the metalnanoparticles by the organic ligands is at least 4.2 ligands per nm².30. The method of claim 21, wherein the organic ligands each includes asulfhydryl group and an amino group as the binding moieties.
 31. Themethod of claim 21, wherein the organic ligands each further includes atleast one hydrophilic moiety.
 32. The method of claim 31, wherein thehydrophilic moiety is a carboxyl group.
 33. The method of claim 21,wherein the solvent is water or heavy water (D₂O).
 34. The method ofclaim 21, wherein the solvent is water.
 35. The method of claim 21,wherein the nanoparticles have an average size in a range of 0.5 nm to10 nm.
 36. The method of claim 21, wherein the metal nanoparticles areimmobilized on a solid support.
 37. The method of claim 21, wherein themetal nanoparticles are bound to larger-sized particles.
 38. The methodof claim 21, wherein removing the ligand-capped nanoparticles isperformed by filtration.
 39. A method for medical imaging, comprising:performing the method for inducing hyperpolarization from para-hydrogenof claim 1; conveying the polarized substrate to a patient; andperforming in vivo imaging of the polarized substrate conveyed to thepatient.
 40. The method of claim 39, wherein the in vivo imagingcomprises MRI.